Despite decades of research, the prevalence of Type 2 diabetes is increasing at epidemic proportions. Our long-term goal is to identify the molecular mechanisms that lead to metabolic dysfunction and insulin resistance. In this application, we will address these challenges, building upon exciting data from our lab that reveals a novel molecular mechanism in controlling adipocyte biology. We have discovered a novel mechanism that contributes to insulin resistance - triggered by loss of SIRT4, a mitochondrial sirtuin. SIRT4 deacetylates and inhibits malonyl CoA decarboxylase (MCD) to control the balance of lipid synthesis and fat oxidation. Consequently, SIRT4KO mice display excessive fat oxidation, and lowered lipid synthesis. Strikingly, though lean, loss of SIRT4 leads to increased insulin resistance. This work elucidates SIRT4 as an important, new, regulator of lipid homeostasis, identifies MCD as a novel SIRT4 target, and deepens our understanding of the malonyl CoA regulatory axis. Our overall hypothesis is that SIRT4 regulates the balance in mitochondrial oxidative metabolism and lipid homeostasis through the modification of MCD in adipocytes, leading to the contribution of excessive mitochondrial flux, decreased adiponectin secretion and insulin resistance. This proposal will test these ideas by our strategy of utilizing biochemical techniques and novel mouse models in the following Aims: 1) To define the enzymatic activity of SIRT4 and identify the molecular mechanism(s) by which SIRT4 represses MCD to regulate lipid homeostasis, 2) To examine SIRT4 and MCD control of oxidative mitochondrial metabolism in adipocyte biology, and elucidate the role of this node in adiponectin production, and 3) To investigate SIRT4- mediated regulation of metabolism and insulin resistance during metabolic challenge by employing novel tools and methods, including fat-specific SIRT4 knockout mice. The innovation of this proposal lies in both the novel tools and the unique hypothesis that SIRT4, a mitochondrial sirtuin, can regulate multiple substrates to control mitochondrial flux and adipokine secretion in adipocytes to contribute to obesity and insulin resistance. The significance of this study is that we will define a molecular pathway in adipocytes that regulates insulin resistance independently of obesity. We will also probe the enzymatic activity and ability of SIRT4, a mitochondrial sirtuin, to promote mitochondrial metabolism, normal adipokine secretion and insulin sensitivity. Success in these experiments would open the door for a new generation of diagnostic methods and pharmacological therapies for treating insulin resistance that would be applicable to many metabolic syndromes.